Libanopsinae, New Subfamily of the Family Sphindidae (Coleoptera

Cretaceous Research xxx (2014) 1e19

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Cretaceous Research

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Libanopsinae, new subfamily of the family Sphindidae (Coleoptera, Cucujoidea) from Lower Cretaceous Lebanese amber, with remarks on using confocal microscopy for the study of amber inclusions

Alexander G. Kirejtshuk a,b,*, Philipp E. Chetverikov a,c, Dany Azar d,b a Zoological Institute of the Russian Academy of Sciences, Universitetskaya emb. 1, St. Petersburg 199034, Russia b CNRS UMR 7205, Muséum National d’Histoire Naturelle, CP 50, Entomologie, 45, rue Buffon, F-75005 Paris, France c Department of Invertebrate Zoology, Saint-Petersburg State University, Universitetskaya emb. 7/9, 199034 St. Petersburg, Russia d Lebanese University, Faculty of Sciences II, Department of Natural Sciences, Fanar, El-Metn 26110217, Lebanon article info abstract

Article history: The present study provides a description of a new subfamily Libanopsinae subfam. nov. with the new Received 19 January 2014 genus Libanopsis gen. nov. and ﬁve new species (L. poinari sp. nov., L. impexa sp. nov., L. limosa sp. nov., Accepted in revised form 14 February 2014 L. straminea sp. nov., L. slipinskii sp. nov.) from Lower Cretaceous Lebanese amber. This extinct subfamily Available online xxx is rather isolated from other subfamilies of the family Sphindidae and shares many external structural features with the extant families Sphindidae and Protocucujidae). The systematic position of the new Keywords: fossils is discussed and key to species is proposed. It was shown that the confocal laser scanning mi- Coleoptera croscopy (CLSM) is a powerful method for surface reconstruction and studying tiny elements of bio- Sphindidae New subfamily inclusions in amber (especially arthropod exoskeletons). The quality of CLSM images drastically depends New genus and species on the medium used for mounting specimens: epoxy resin totally masks the signal from the inclusion Lebanese amber under examination, whereas samples prepared Canada Balsam are highly appropriate for CLSM study. Lower Cretaceous Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction placed in taxa proposed for the Recent fauna. Thus the fossil record of this superfamily (especially its Mesozoic part) is unreliable for The superfamily Cucujoidea embraces the greatest number of phylogenetic reconstructions. A more detailed overview of cucu- families within the superfamilies of the order Coleoptera. Bouchard joids in the fossil record is available in the catalogue of Kirejtshuk et al. (2011) listed 35 recent and 2 extinct families in this group, and Ponomarenko (2013). however the numbers are disputed with various specialists, having This paper describes a new Mesozoic sphindid subfamily their own interpretations. Also most present-day researchers (Libanopsinae subfam. nov.) from Lower Cretaceous Lebanese distinguish many small separate families with comparatively amber. This fossil group has clear connection with members of the minute diagnostic characters (Leschen et al., 2005 etc.) and the Recent fauna and could be very significant in illustrating the authors of the present paper feel obliged to follow this tradition. Mesozoic diversification of the superfamily Cucujoidea. This new Great complexities exist in the interpretation of fossils which, on subfamily of the Sphindidae Jacquelin du Val, 1858 is represented one hand, are still rather poorly known in general, especially those by a new genus, Libanopsis gen. nov., with five new species: from the Mesozoic. On the other hand, the most reliable records L. poinari sp. nov., L. impexa sp. nov., L. limosa sp. nov., L. straminea from this era are mostly from the few Cretaceous amber inclusions. sp. nov., L. slipinskii sp. nov., which constitute the earliest record of Unfortunately small cucujoids are comparatively rare in compres- the family. sion deposits and are often not well preserved, making them difficult to study and compare with recent representatives. As a 2. Material and methods result, uncommon extinct species and groups were tentatively The Lebanese amber specimens examined are deposited in the Lebanese University, Faculty of Sciences II, Department of Natural Sciences. For this study, basic optical equipment was used, * Corresponding author. Zoological Institute of the Russian Academy of Sciences, Universitetskaya nab. 1, St. Petersburg 199034, Russia. including a stereomicroscope (Leica MZ 16.0) in the Saint Peters- E-mail addresses: [email protected], [email protected] (A.G. Kirejtshuk). burg Institute and a stereomicroscope (Olympus SCX9) in the Paris http://dx.doi.org/10.1016/j.cretres.2014.02.008 0195-6671/Ó 2014 Elsevier Ltd. All rights reserved.

Museum. In addition, the specimens were studied with a confocal dimensional representation of the insect surface topography (Iso- laser scanning microscope in the Saint Petersburg State University surface and Volume rendering images) were carried out using Ò Ò (see below). The method of preparation of the specimens for study Amira 5.3.2 software according Amira User’s Guides based on the was described in Azar et al. (2003). confocal stacks obtained from the single insect specimen that provided the best image. All 3-dimensional images were recorded Strata. Lower Cretaceous; ante mid-Barremian (circa 130e145 Myr), using “PrintScreen” keyboard function or “Snapshot” command Ò after the most recent geological and stratigraphical data (unpub- embedded in Amira . All images were checked for brightness and Ò lished, in preparation). contrast correction with Photoshop CS11 version 12.1 (Adobe Systems Inc.). Localities. Specimens 841 BC and 474 B originated from the Ham- mana/Mdeyrij outcrop [Caza (Department) Baabda, Mouhafazet 3. Systematic paleontology Jabal Loubnan (Mount Lebanon Governorate)], Central Lebanon (Fig. 1); specimen J-6F from the Bkassine (Jouar Ess-Souss) outcrop Order Coleoptera Linnaeus, 1758 [Caza (Department) Jezzine, Mouhafazet Loubnan El-Janoubi Infraorder Cucujiformia Crowson, 1960 (South Lebanon Governorate)], Southern Lebanon (Fig. 2); spec- Superfamily Cucujoidea Latreille, 1802 imen TAR-101A from the Bouarej outcrop [Caza (Department) Family Sphindidae Jacquelin du Val, 1858 Zahleh, Mouhafazet El Beqaa (Beqaa Governorate)] Central Lebanon Subfamily Libanopsinae Kirejtshuk, subfam nov. (Fig. 1); and specimen NBS-4C from the Nabaa-Ess-Sukkar/Brissa Type genus Libanopsis gen. nov. outcrop [Caza (Department) Danniyeh, Mouhafazet Loubnan Esh- Shemali (North Lebanon Governorate) Northern Lebanon (Fig. 3).

Diagnosis. Body rather small (length 1.1e1.8 mm); subelliptic, Confocal laser scanning microscopy (CLSM) moderately convex dorsally and subflattened ventrally; dorsum dark brownish to blackish; underside brown to blackish; append- For CLSM studies, polished blocks of amber with specimens ages usually lighter; mostly mat; dorsum with sparse, stout and embedded in epoxy resin and Canada Balsam were used. The CLSM erect hairs; underside with fine and moderately dense pubescence. procedure was carried out in the Center of Microscopy and Dorsum diffuse, coarsely and sparsely punctured, with smooth Microanalysis at Saint-Petersburg State University using the Spec- microsculpture. Underside with fine and moderately dense punc- tral confocal & multiphoton system of Leica TCS SP2 with objectives tures with apparently smooth sculpture between them. Head large, 63 N.A. 1.4-0.60 Oil lBL HCX PL APO and 40 N.A. 1.25-0.75 Oil CS more or less short, subtriangularly transverse, somewhat convex HCX PL APO at an excitation wavelength of 405 nm (blue laser, 50% dorsally, more or less declined ventrally, with large eyes; eyes intensity), and an emission wavelength range of 415e750 nm. The coarsely facetted (on underside as large as on dorsum); antennal most common acquisition resolution was 2048 2048 pixels, level insertions located just before anterior edge of eyes. Labrum uni- of gain 550e650, frame average 1 or 2, and zoom range of 1.5e3.0 lobed and slightly exposed from under anterior edge of frons. times. Between 30 and 65 optical slices were recorded from each Mandibles apparently moderately developed, gradually curved specimen examined. The digital images of the confocal stacks were externally and with bidentate apices. Maxillae apparently with one processed using Fiji Open Source Image Processing Package to lobe (?galea) setose at apex and 4-segmented palpi. Labial palpi 3- obtain the maximum intensity projections (MIP). Three- segmented. Antennae 10-segmented, moderately long, with

Fig. 1. Geological map of Hammana, and Bouarej amber outcrops. Abbreviations: J6 ¼ Kimmeridgian; bJ6 ¼ Volcanic Kimmeridgian; C1 ¼ Neocomian e Ante Barremian; bC1 ¼ Volcanic Neocomian; C2a ¼ Barremian e ante mid Barremian; C2b ¼ End Barremian e Aptian; C3 ¼ Albian; C4 ¼ Cenomanian; Q ¼ Quaternary; thickened lines represent faults; scale bar ¼ 1 km. Purple arrow indicates the Hammana outcrop; red arrow indicates the Bouarej outcrop. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

Fig. 2. Geological map of Bkassine outcrop. Abbreviations: J6 ¼ Kimmeridgian; J7 ¼ Tithonian; C1 ¼ Neocomian e Ante Barremian; C2a ¼ Barremian e ante mid Barremian; C2b ¼ End Barremian e Aptian; C3 ¼ Albian; C4 ¼ Cenomanian; Q ¼ Quaternary; thickened lines represent faults; scale bar ¼ 1 km. White arrow indicates the amber outcrop.

Fig. 3. Geological map of Nabaa Ess-Sukkar/Brissa outcrop. Abbreviations: C1 ¼ Neocomian e Ante Barremian; C1-2 ¼ Neocomian e ante mid Barremian; C2b ¼ End Barremian e Aptian; bC2b ¼ Volcanic Aptian; C3-4 ¼ Albian e Cenomanian; C4 ¼ Cenomanian; Q ¼ Quaternary; thickened lines represent faults; scale bar ¼ 1 km. Yellow arrow indicates the amber outcrop. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

enlarged antennomeres 1 and 2, and also with 3 apical anten- not reaching elytral epipleura. Abdomen with five ventrites. Epi- nomeres forming a loose club. Pronotum transverse, gently vaulted pleura of elytra moderately narrow and gradually narrowing and with arcuate sides undulated (to crenulated) at edges. apically, distinctly slopping externally. Legs narrow and long. Tro- Scutellum subtransverse, widened and subtruncate at apex. Elytra chanters of elongate to heteromeroid type and moderately short. complete, somewhat convex along middle and steeply sloping at Tibiae rather thin, without spurs. Femora of usual configuration. narrowly subexplanate sides, adsutural lines well expressed at least Tarsi 5-5-5, with tarsomeres 1e4 more or less lobed and ultimate along distal half of suture. Abdomen invisible dorsally. Fifth tarsomere longest, claws simple or subdentate at base. maxillary palpomere slightly subconical and somewhat narrowed at base, elongate third labial palpomeres subconical and sub- Comparison. truncate at narrow apex. Antennal grooves developed. Prosternum with process moderately wide (markedly narrower than procoxal This new subfamily was mentioned in Kirejtshuk and Azar cavities and moderately far projecting beyond posterior edge of (2013) as family “somewhat resembling Calitys Thomson, 1859 procoxae). Procoxae transversely oval and open posteriorly. Dis- but with oval and open posteriorly procoxae and long erect setae on tance between oval mesocoxae comparable to that between pro- dorsum”. In addition to the peculiar structure of procoxae, the coxae. Mesoventrite about half as long as prosternum and adults are characterised by the 5-segmented tarsi of all legs in apparently not depressed in respect to plane prosternum and combination with the 5-segmented abdomen, complete elytra and metaventrite. Metaventrite slightly convex along midline. Mete- unmodified mouthparts. These peculiarities provide an opportu- pisterna narrow. Metacoxae narrowly separated or conjointed and nity to compare this group among the superfamilies Cleroidea and

Fig. 4. Libanopsis poinari sp. nov., holotype “841 BC”:Ae body, dorsal view; B e idem, ventral view; C e anterior part of body, ventral view. Body length 1.4 mm.

Cucujoidea of the infraorder Cucujiformia because the groups of the with 10-segmented clubbed antennae, procoxae widely open pos- superfamily Tenebrionoidea have not imaginal tarsi 5-5-5 and teriorly, ﬁve abdominal ventrites and tarsi 5-5-5 in both sexes occur those of Lymexyloidea demonstrate more or less modiﬁed in Lithophilini Imhoff, 1856 (Coccidulinae Mulsant, 1850: Cocci- mouthparts (particularly maxillary palpi). Instead of showing some nellidae Latreille, 1807), Hypocoprini (Atomariinae LeConte, 1861: external similarity to some cleroids such as Trogossitidae Latreille, Cryptophagidae Kirby, 1837) and Sphindinae (Sphindidae). How- 1802, sensu lato (including Peltidae Latreille, 1807 and Lophoca- ever, the new species are quite distinct from the members of both teridae Crowson, 1964); Phloiophilidae Kisenwetter, 1863, the genera Tetrabrachys Kapur, 1948 (Lithophilini) and Hypocoprus adults of this new subfamily have no remains of tarsal empodia and Motschulsky, 1839 (Hypocoprini) due to their large and coarsely their metacoxae do not reach the elytral epipleura, therefore, they faceted eyes, frontal elevations over the antennal insertions, rather can be scarcely placed with the cleroids. At the same time, adults different structure of the antennae, pronotum with undulate edges

Fig. 5. Species of Libanopsis gen. nov.: A e body of holotype of L. poinari sp. nov., “841 BC”, dorsal view; B e anterior part of body of the same specimen, ventral view; C e body of holotype of L. impexa sp. nov., “474 B”, dorsal view. Body length of holotype of L. poinari sp. nov. 1.4 mm. Body length of holotype of L. impexa sp. nov. 1.3 mm.

Please cite this article in press as: Kirejtshuk, A.G., et al., Libanopsinae, new subfamily of the family Sphindidae (Coleoptera, Cucujoidea) from Lower Cretaceous Lebanese amber, with remarks on using confocal microscopy for the study of amber inclusions, Cretaceous Research (2014), http://dx.doi.org/10.1016/j.cretres.2014.02.008 6 A.G. Kirejtshuk et al. / Cretaceous Research xxx (2014) 1e19 somewhat similar body shape and undulate lateral edges of the Holotype. “841 BC”, probable female; complete specimen; inclusion pronotum (Sphindus Dejean, 1821), subquadrangular scutellum and in a thin subquadrangular piece of more or less homogeneous light rather sparse and coarse punctation of the dorsum (Notosphindus amber (3.1 mm long and 2.0 mm wide) with some pieces of McHugh and Wheeler, 1991 but with another shape), and tarsi 5-5- brownish organic matter in front of the specimen and at its distal 5 in both sexes (Forrester, 2003: different species of Aspidiphorus part. The piece of amber was prepared and embedded between two Sturm, 1826 have males with both tarsal formula 5-5-4 and 5-5-5). round microscope cover slips in a Canada Balsam medium. The The species of the genus Eurysphindus LeConte, 1878 shows the dorsum and underside of the specimen are covered with a thin epipleura subhorizontal with outer parts slightly declined down- layer of air making it difficult to observe outlines of scleritesa and wards (somewhat similar to those in species of the new genus), the sculpture of the integument. subexplanate pronotal sides with undulate edges, diffuse punctation of elytra, thick suberect pubescence on dorsum and more or Diagnosis. This new species differs from the congeners in the less narrowly separated pairs of coxae. The sphindid groups with characters mentioned in the key to species below. In addition, this 11-segmented antennae also show some similarities with species of new species has a more slender body, the head somewhat longer the new genus. Particularly, the subfamily Protosphindinae, which before the eyes and more flattened dorsally in comparison with have according to Sen Gupta and Crowson (1979) the “sides of frons L. impexa sp. nov., also the eyes are larger, some ratios of the markedly raised, partly hiding antennal insertions”, although, in antennomeres differ, the top anterior angles of the pronotum are contrast to Libanopsis species, they bear other different diagnostic less distinct, the elytra apices are more widely rounded and it has a features (listed above), especially very different sculpture and longer prosternum. Libanopsis poinari sp. nov. also differs from punctation of the elytra (not sparse and diffuse coarse puntures) L. limosa sp. nov. in having dark setae on the dorsum, apparently and lack of thick setae on the dorsum. less elevated interspaces between punctures on the elytra, a The new sphindid subfamily differs from the apparently closest thinner base of tarsomere 1 and some proportions of the cucujoid group, the Protocucujidae Crowson, 1954, in the absence antennomeres. of well developed bisetose empodia, non flattened antennal club, the frons completely covering the base of the antennae and forming Description of holotype. Body length 1.4 mm, width 0.5 mm, elon- wide lateral lobes (in species of the genus Ericmodes Reitter, 1878 gate, rather convex dorsally and subflattened ventrally; dorsum the frons only slightly cover the antennal bases), 10-segmented dark brown to black; underside dark brown; appendages reddish antennae, anterior part of prothorax strongly extended anteriorly, with lighter tarsi and dark tarsal claws; dorsum with uniform, procoxae widely open posteriorly, subcontiguous metacoxae, epi- rather long and sparse, thick erect brownish hairs (setae) some- pleura sloping externally, male tarsi 5-5-5. what longer than distance between their insertions; underside The confocal microscopy gave opportunity to study the apparently with fine, short and moderately dense hairs. mouthparts of the specimens under description in more detail than Dorsum with subuniform, rather large, diffuse and rather sparse it is possible due to usage of common optics for study of amber suboval punctures with diameter about twice as great as that of inclusions. They are more or less similar to those in recent members facets; interspaces between them more than two puncture di- of Sphindidae and Protocucujidae, however, as it is seen on Fig. 6C ameters, probably more or less smoothed; interspaces between the maxilla of Libanopsis poinari sp. nov. looking like bearing only punctures on elytra rather elevated, forming irregular smooth one lobe, but not two ones as known in the recent groups of the ridges. Underside apparently with subuniform very fine and sparse mentioned families (McHugh and Wheeler, 1991; Slipinski, 1999; punctation and somewhat smoothed interspaces. Forrester and McHugh, 2007). If this difference between the fossil Head subflattened dorsally and apparently with transverse Libanopsinae subfam. nov. and recent Sphindidae is really stable it impression at the level of eye anterior edges and with slight ele- could be regarded as an additional trace of rather ancient diver- vations over antennal insertions; anterior edge of frons transverse gence of these groups (see the below discussion). and from under it a moderately short transverse and heavily sclerotised labrum is apparently exposed. Eyes rather large, coarsely Genus Libanopsis Kirejtshuk, gen. nov. facetted and globular, distance between them about 4 times as great as eye transverse diameter. Antennae moderately long and Type species. Libanopsis poinari sp. nov. not reaching the posterior edge of pronotum; antennomere 1 (scape) rather large and subglobular; antennomere 2 large and sublobular, somewhat smaller than scape and somewhat longer Derivation of name. The name of this new genus is derived from the than wide; antennomere 3 narrow and longest in flagellum, Greek name of country of origin “Liban” and from the Greek “opsis” apparently comparable in length with scape and slightly shorter meaning “resembling a (specified) thing”. than ultimate antennomere; both antennomeres 4 and 6 slightly shorter than antennomere 5 which in turn is shorter than anten- Composition. Besides the type species, the new genus includes nomere 3; antennomere 7 shortest and thicker than antennomeres L. impexa sp. nov., L. limosa sp. nov., L. slipinskii sp. nov. and 3e6 at apex (subconical), antennomeres 8e10 forming a loose and L. straminea sp. nov. slightly dorsoventrally compressed club which is markedly thicker than scape and comprising about 2/5 of total antennal length; ul- Diagnosis. The same as for the new subfamily. timate antennomere apparently somewhat longer than scape and about 1.5 times as long as each of antenomeres 8 and 9. Pronotum Libanopsis poinari Kirejtshuk, sp. nov. about 1 and 1/3 as wide as long with subexplanate sides about as Figs. 4; 5 A, B; 6; 7 A, B wide as antennal club, moderately convex at disk, evenly arcuate along sides, widest at the middle, lateral edges moderately undulate, with anterior and posterior angles not projecting, posterior Derivation of name. The epithet of this new species is dedicated to ones nearly rounded and anterior ones with a distinct top. George Poinar Jr. who cooperated with the senior author of this Scutellum strongly transverse, somewhat widening posteriorly and paper during many years and assisted him with scientific and lin- about twice as wide as long. Elytra complete, elongate, widest at guistic matters. base, almost twice as long as wide combined and nearly three times

Fig. 6. CLSM images (A e Isosurface, BeF e maximum intensity projections) of Libanopsis poinari sp. nov., holotype “841 BC”:Ae body, dorsal view (body length 1.4 mm); B e anterior part of body, ventral view; C e mouthparts, ventral view; D e apex of mesotibia and mesotarsomere 1, ventral view; E e anterior tarsal claws, ventral view; F e mesotarsus, dorsal view. Abbreviations: c e cardo, li e ligula, lp e labial palpus, md e mandible, ml e maxillary lobe, mp e maxillary palpus. as long as pronotum, subparallel-sided and their apices forming a Underside of head with distinct elevation along middle behind widely rounded joint arc, rather convex and steep at sides, lateral mentum. Hypostomal sinuses moderately deep with well devel- edge slightly undulate. Pygidium with widely rounded apex and oped maxilla bearing 5-segmented palpus of usual structure and slightly exposed from under apices of elytra. ultimate palpomere slightly subconical and apparently more than

Fig. 7. CLSM images (A,B,D e maximum intensity projections; C e volume rendering) of species of Libanopsis gen. nov. : A e prosternum and mesoventrite of holotype of L. poinari sp. nov., “841 BC”, ventral view; B e maxillary and labial palpi of the same specimen, ventral view; C e body of holotype of L. slipinskii sp. nov., “NBS 4C”, dorsal view; D e body of holotype of L. impexa sp. nov., “474 B”, dorsal view (body length 1.3 mm). Abbreviations: lbr e ligula, lp e labial palpus, mc e mesocoxa; md e mandible, mp e maxillary palpus, pc e procoxa, pp e prosternal process.

three times as long as thick. Mentum subpentangular, four times as apparently longest, ventrite 2 somewhat shorter, ventrites 3e5 wide as long. Labial palpi 3-segmented and with ultimate palpo- apparently comparable in length and hypopygidium widely mere subcylindrical and about 2.5 times as long as wide. Pros- rounded at apex. Epipleura at anterior third of elytra rather wide ternum gently and narrowly convex along the middle with rather (somewhat wider than antennal club) and gradually narrowing and long and narrow intercoxal process. Procoxae not widely trans- becoming obsolete at apex. verse. Procoxal cavities widely open posteriorly. Pronotosternal Legs moderately long and narrow. All trochanters of hetero- sutures slightly evident at outer end of procoxal cavities. Distances meroid rather than elongate type, particularly metatrochanter. between pro- and mesocoxae obvious. Mesoventrite moderately Femora of usual shape, narrow and comparable in shape, pro- and short, about half as long as prosternum, apparently of usual mesofemora about 3.0 times as long as wide, metafemur about 2.5 structure. Mesocoxae subtriangularly oval. Metaventrite slightly times as long as wide. Tibiae narrow, about as long as femora and convex and with median excavation at its posterior edge between slightly widening apically. Tarsi with four ﬁrst tarsomeres with one metacoxae. Metepisterna subtriangular and widened anteriorly. lobe; tarsomere 1 somewhat longer than each of tarsomeres 2e4; Metacoxae transverse. Abdomen with ﬁve ventrites; ventrite 1 tarsomere 5 slightly longer than tarsomeres 2e4 combined and very

Fig. 8. CLSM images (A,C e maximum intensity projections; B e Isosurface, Orthoslice and volume rendering combined image; D e volume rendering) of Libanopsis impexa sp. nov., holotype, “474 B”:A,Be body, ventral view; C e posterior leg, ventral view; D e pro- and mesotarsi, ventral view. Abbreviations: mf e metafemur, mt e metatibia, mtr e metatarsus.

thin at base and at apex markedly more than twice as wide as at prepared and embedded between two round microscope cover base; claws rather long and slightly swollen (with serration) at base. slips in a Canada Balsam medium. The dorsal integument of the specimen is more or less clear along the centre but with some Libanopsis impexa Kirejtshuk, sp. nov. “milky” covering and apparently organic debris along its sides; its underside is rather obscured with some “milky” deposit, although Figs. 5 C; 7 D; 8; 9; 10 A the head and part of thoracic sclerites are visible.

Derivation of name. The epithet of this species, the Latin “impexus” Diagnosis. This new species is similar to L. poinari sp. nov., but dif- means bald, simple, coarse, unvarnished, unembellished, un- fers from it and all congeners in the characters mentioned below in combed; tousled. the key to species. In addition, see the diagnosis of the previous species and following species. Holotype. “474 B”, sex unclear; complete specimen; inclusion in a thin subquadrangular piece of more or less homogeneous light Notes. Taking into consideration the great similarity of this new amber (3.4 mm long and 1.6 mm wide). The piece of amber was species with the type species of the genus, common characters

Fig. 9. Libanopsis impexa sp. nov., holotype, “474 B”:Ae body, dorsal view; B e idem, ventral view; C e metaventrite and abdomen, ventral view. Body length 1.3 mm.

Please cite this article in press as: Kirejtshuk, A.G., et al., Libanopsinae, new subfamily of the family Sphindidae (Coleoptera, Cucujoidea) from Lower Cretaceous Lebanese amber, with remarks on using confocal microscopy for the study of amber inclusions, Cretaceous Research (2014), http://dx.doi.org/10.1016/j.cretres.2014.02.008 A.G. Kirejtshuk et al. / Cretaceous Research xxx (2014) 1e19 11 scape and slightly longer than ultimate antennomere; anten- (male anal sclerite) with long setae, which precludes the genital nomere 4 similar to antennomere 3 but somewhat shorter; capsule from being retracted inside the abdomen. antennomeres 5 and 6 similar and each of them slightly shorter than antennomere 4; antennomere 7 as long as each of two pre- Description of holotype. Body length 1.2 mm, width 0.6 mm, height vious ones and much thicker at apex, antennomeres 8e10 forming 0.3 mm, elongate, rather convex dorsally and subflattened a loose and slightly dorso-ventrally compressed club which is ventrally; dorsum blackish brown with dark (dark brown) ap- somewhat wider than scape and about 1/3 of entire antennal pendages; with uniform, rather long and sparse, comparably thin length; ultimate antennomere subequal in length with scape and and erect whitish hairs (setae) somewhat longer than distance about 1.5 times as long as each of antennomere 8 and 9. Pronotum between their insertions; underside apparently with fine, short and about 1 and 2/3 as wide as long with subexplanate sides that are moderately dense hairs (slightly visible on prosternum and distal wider than antennal club, moderately convex at disk, evenly part of abdomen). Interspaces between punctures on elytra more arcuate along sides, widest at middle, posterior and anterior ones than two puncture diameters, probably more or less smooth; in- with a distinct top. Elytra complete, elongate, widest at base, terspaces between punctures on elytra slightly vaulted. together about 1 and 3/5 as long as wide and somewhat more than Head apparently subflattened dorsally with slight elevations three times as long as pronotum, subparallel-sided and their apices over antennal insertions; anterior edge of frons transverse. Eyes forming a joint arc. Pygidium with widely rounded apex and rather large, coarsely facetted and globular, distance between apparently slightly exposed from under apices of elytra. them about 4 times as great as eye transverse diameter. Antennae Underside of head subflattened behind mentum. Prosternum moderately long and not reaching the posterior edge of prono- gently convex and wide along the middle with rather long and tum; antennomere 1 (scape) moderately large and slightly com- narrow intercoxal process. Pronotosternal suture clearly visible pressed dorsoventrally; antennomere 2 large and slightly between outer end of procoxal cavities. Distances between pro- and compressed dorsoventrally, somewhat smaller than scape and mesocoxae comparable. Mesoventrite slightly shorter than pros- longer than wide; antennomere 3 narrow and longest in flagel- ternum. Metaventrite rather convex. Abdomen with five ventrites; lum, longer than scape and longer than ultimate antennomere; ventrites 1 apparently longest, ventrite 2 somewhat shorter, ven- each of antennomeres 4e6 rather short; antennomeres 8e10 trites 3 and 4 apparently comparable in length and hypopygidium forming a loose and slightly dorsoventrally compressed club somewhat longer than two previous ones and widely rounded at which is somewhat thicker than scape and composing about one apex. Epipleura at anterior third of elytra rather wide (about twice third of the total antennal length; ultimate antennomere some- as wide as antennal club). what shorter than scape and about 1.5 times as long as each of Tarsi with four first tarsomeres with one lobe; tarsomere 1 antennomeres 8 and 9. Pronotum almost twice as wide as long somewhat longer than each of tarsomeres 2e4; tarsomere 5 with very narrow subexplanate sides (much less than width of slightly longer than tarsomeres 2e4 combined, at base only slightly antennal club, rather convex at disk, evenly arcuate along sides, narrower than at apex. widest at basal third, with anterior and posterior angles not projecting and with a rounded top. Scutellum not visible. Elytra Libanopsis limosa Kirejtshuk, sp. nov. complete, elongate, widest at base, each elytron less than 3 times (nearly 2.5 times) as long as wide and apparently three times as Figs. 10 B, C; 11 long as pronotum, broadly arcuate with their apices forming a widely rounded joint arc, rather convex and steep at sides, lateral Derivation of name. The epithet of this species refers to the dark and edge slightly undulate. subdued colouration of this new species masking by thick layer of Underside of head apparently flattened behind mentum. Ulti- “milky cover”, the Latin “limosus” means muddy, dirty, miry, silty, mate maxillary palpomere slightly subconical and apparently more oozy, shady. than three times as long as wide. Mentum and labial palpi invisible. Prosternum gently and narrowly convex along the middle with Holotype. “J-6 F”, probable male; specimen missing the distal part of rather long and narrow intercoxal process. Distances between the left antenna and somewhat broken distal part of the elytra; procoxae somewhat greater than that between mesocoxae. Meta- inclusion in a thin subquadrangular piece of more or less homo- ventrite and metepisterna apparently as in two previous species. geneous light amber (2.2 1.2 1.0 mm). The piece of amber was Abdomen with ventrites 1 longest, ventrite 2 somewhat shorter prepared and embedded in parallelogram bar (17.2 4.0 2.2 mm) than ventrite 1, ventrites 3e5 apparently comparable in length with with Canada Balsam medium. Part of the dorsum and most of the hypopygidium rather widely rounded to subtruncate at apex. Epi- underside of the specimen are covered with a thick layer of “milky pleura gradually narrowing as in two previous species. Heavily deposit” and most of the remaining integument is scarcely visible sclerotised anal sclerite broken on one side and rather exposed because of various organic matter above it. from under segment VII. Exposed parameres with apices curved medially. Diagnosis. This new species differs from its congeners in the char- Femora and tibiae of usual shape. Tarsi with four first tarso- acters mentioned in the key to species below. Also, see the di- meres with one lobe; tarsomere 1 somewhat longer than each of agnoses of the previous and following species. Libanopsis limosa sp. tarsomeres 2e4; tarsomere 5 slightly longer than tarsomeres 2e4 nov. differs from two previous species in the somewhat more combined and at base only slightly thinner than at apex; claws convex pronotum with very narrow subexplanate sides and widest rather long and dentate at base. portion in the basal third. The two following new species, in contrast to the two previous ones and L. limosa sp. nov., are Libanopsis straminea sp. Kirejtshuk, nov. markedly more robust. Fig. 12 Notes. Taking into consideration the great similarity of this new species with the type species of the genus, common characters Derivation of name. The epithet of this species refers to the col- shared by both are omitted in the description below. The specimen ouration of this species; the Latin “stramineus” means straw has a genital capsule exposed from under abdominal segment VII coloured.

Fig. 10. Species of Libanopsis gen. nov.: A e head and prothorax of holotype of L. impexa sp. nov., “474 B”, ventral view; B ebody of L. limosa sp. nov., “J-6 F”, ventral view; C e idem, lateral view. Body length of holotype of L. impexa sp. nov. 1.3 mm. Body length of holotype of L. limosa sp. nov. 1.2 mm.

Holotype. “TAR-101 A”, probable female; specimen with exposed Diagnosis. This new species differs from the congeners in the pieces of both hindwings and with missing portion of pronotum characters mentioned in the below key to species. Besides, this new and dorsal side of head; inclusion in a thin subquadrangular piece species differs from congeners in the somewhat larger and lighter of homogeneous light amber (3.1 mm long and 4.0 mm wide) body and appendages, two types of setae on dorsum, probably very with small dark wood filament at base of left elytron and fine shallow punctures on dorsum. This new species has the dorsum threads of organic matter at its apex (one thread long and ori- clearly more convex than that in L slipinskii sp. nov. Finally, the ented obliquely adjacent to the specimen. There are also some integument of base of head, prosternum and metaventrite of layers of organic matter along the right side of the distal part of L. straminea sp. nov. rather smooth and without visible pubescence. the specimen. The piece of amber was prepared and embedded between two square microscope cover slips in Canada Balsam. Notes. Taking into consideration the great similarity of this new The dorsum of the specimen is covered with a thin layer of air species with the type species of the genus, most characters shared making it scarcely possible to observe outlines of sclerites and by both are omitted in the description below. sculpture patterns of the integument; the underside has a thick “milky” covering on the head, distal part of prothorax, meso- Description of holotype. Body length 1.8 mm, width 0.7 mm, elon- thorax and abdomen. gate oval, moderately convex dorsally and subflattened ventrally;

Fig. 11. Libanopsis limosa sp. nov., holotype “J-6 F”:Ae body, ventral view; B, C e idem, lateral view; D e abdomen, ventral view. Body length 1.2 mm. dorsum dark brown; underside and appendages reddish; dorsum Antennae moderately long and not reaching the posterior edge with rather long and sparse and thick brownish hairs (setae), of pronotum; antennomere 1 (scape) rather large and subglobular; among which are some erect setae; some arcuate setae are curved antennomere 2 large and elongate oval, somewhat smaller than in the middle and orientated toward the surface of the integument, scape and somewhat longer than wide; antennomere 3 rather these setae are somewhat longer than the distance between their narrow and the longest in the ﬂagellum, apparently comparable in insertions; underside without visible hairs. length with the scape and slightly shorter than ultimate anten- Dorsum apparently with subuniform, large, diffuse and rather nomere; each of antennomeres 4e6 half as long as antennomere 3; sparse suboval large punctures which could be shallow and indis- antennomere 7 somewhat longer and at apex wider than anten- tinct; sculpture on interspaces between punctures invisible. Visible nomeres 3e6 (subconical) but shorter than antennomere 3, integument of head base, prosternum and metaventrite without antennomeres 8e10 forming a loose and slightly dorso-ventrally clear punctation, smooth. compressed club which is about twice as wide as scape and

Fig. 12. Libanopsis straminea sp. nov., holotype “TAR 101 A”:Ae body, dorsal view; B e idem, ventral view; C e anterior part of body, ventral view. Body length 1.8 mm. comprising about 2/5 of total antennal length; ultimate anten- Prosternum gently and slightly convex along the middle and nomere apparently somewhat longer than scape and about 1.5 with intercoxal process slightly widened at truncate apex. Prono- times as long as each of antenomeres 8 and 9. Pronotum widest in tosternal sutures not visible. Distances between procoxae about as posterior third, with subexplanate and undulate sides about as wide as antennal club. Metaventrite slightly convex. Abdominal wide as antennal club. Scutellum not visible. Elytra complete, ventrites 1 apparently longest, ventrites 2e5 apparently compa- elongate, about 1.5 times as long as wide combined, subparallel- rable in length and hypopygidium widely rounded at apex. Epi- sided in basal 2/3 with their apices forming an angle, moderately pleura at anterior third of elytra rather wide (about as wide as convex and gently sloping, lateral edge slightly undulate. Pygidium antennal club). with widely rounded apex and not exposed from under apices of Pro- and mesofemora apparently more than 3.0 times as long as elytra. wide, metafemur nearly 3.0 times as long as wide. Tarsi with four

Fig. 13. Libanopsis slipinskii sp. nov., holotype “NBS 4C”:Ae body, dorsal view; B e idem, ventral view; C e anterior part of body, ventral view. Body length 1.1 mm.

ﬁrst tarsomeres with one lobe; tarsomere 1 somewhat longer than consultations on the comparison of fossil species examined with each of tarsomeres 2e4; tarsomere 5 slightly longer than tarso- members of the Recent fauna. meres 2e4 combined and very thin at base; at apex about twice as thick as at base; base of claws not visible. Holotype. “NBS-4 C”, probably male; complete specimen; inclusion in a thin subquadrangular piece of more or less homogeneous light Libanopsis slipinskii Kirejtshuk, sp. nov. amber (2.5 mm long and 2.0 mm wide) with some brown pieces of organic matter along the right side of the distal part of the spec- Figs. 7 C; 13; 14 imen. The piece of amber was prepared and embedded between two round microscope cover slips in Canada Balsam. The dorsal Derivation of name. The epithet of this new species is devoted to integument of the specimen is somewhat soiled and its underside Adam S. Slipinski who provided the senior author with numerous bears a layer of “milky deposit” (rather thick at sides) making it

Key to the species

1. Head and pronotum slightly transverse; pronotum at most twice as wide as long ...... 2 - Head and pronotum strongly transverse; pronotum more than three times as wide as long ...... 4 2 (1). Appendages nearly black; body less slender: each elytron less than three times (nearly 2.5 times) as long as wide; pronotum with rather narrowly subexplanate sides; ultimate tarsomere at base slightly narrower than at apex; tarsal claw distinctly dentate at base. Figs. 10 B, C; 11 ...... L. limosa sp. nov. - Appendages brownish to reddish; body more slender; each elytron about three times as long as wide or longer; tarsal claws with a small serration ...... 3 3 (2). Pronotum less than 1.5 times as wide as long with subexplanate sides about as wide as antennal club; elytra regularly subparallel-sided; interspaces between punctures on elytra strongly elevated forming irregular smoothed ridges; underside of head somewhat elevated along the middle; distance between mesocoxae less than width of antennal club; metaventrite longer than prosternum with Fig. 14. Libanopsis slipinskii sp. nov., holotype “NBS 4C”:Ae head and pronotum, intercoxal process; ultimate tarsomere much thinner than at ventral view; B e head and ptothorax, ventral view. Body length 1.1 mm. apex (with difference in thickness almost three times).

Figs. 4; 5 A, B; 6; 7 A, B ...... Protocucujidae. Other characters need further study, although such ...... L. poinari sp. nov. diagnostic characters as number of the antennomeres in antennal club and number of flagellomeres, level of separation of all pairs of - Pronotum more than 1.5 times as wide as long with sub- coxae and so on seem to be a sequence of homoplasy in structural explanate sides wider than antennal club; elytra widest at transformations. distal third; underside of head flattened; interspaces be- The recent cladistic comparison of imaginal and larval characters tween punctures on elytra slightly vaulted; distance between of extant coleopterous groups (Lawrence et al., 2011)showsthat mesocoxae greater than width of antennal club; metaven- cucujoids can be interpreted as four clades with different links and trite and prosternum with intercoxal process subequal in both Sphindidae and Protocucujidae fall in the same clade at the base length; ultimate tarsomere only slightly thinner than at apex. of the infraorder Cucujiformia. The family Protocucujidae in the Figs. 5 C; 7 D; 8; 9; 10 A ...... cladogram of these authors was placed together with Helotidae L. impexa sp. nov. Chapuis; 1876, most part of Erotylidae Latreille; 1802, Byturidae 4 (2). Elytral apices forming nearly a regular arc at apices; body Gistel,1848 and some others. At the same time the family Sphindidae about twice as long as wide; pronotum widest at the middle were placed in another clade together with Hobartiidae Sen Gupta and with subexplanate sides much wider than antennal club; and Crowson, 1966; Boganiidae Sen Gupta et Crowson, 1966; Cryp- antennae longer (reaching posterior edge of prosternum), tophagidae Kirby, 1826; Priasilphidae Crowson, 1973; another part of with thinner antennomeres, and rather loose and scarcely Erotylidae (genus Toramus Grouvelle, 1916); Cucujidae Latreille, dorsoventrally compressed antennal club which is more than 1802; Agapythidae Sen Gupta and Crowson, 1969 and Silvanidae three times as long as wide; most setae of dorsum markedly Kirby, 1837. Thus, the latter clade includes about a fourth of the more than twice as long as interstices between them. Figs. 7 family groups of Cucujoidea and joins forms with very different adult C; 13; 14 ...... L. slipinskii sp. structural characters. A strict analysis of these characters will nov. become possible after the descriptions at least major Mesozoic groups of cucujoids. Hunt et al. (2008), after comparison of some - Elytral apices forming an angle at apices; body about 2.5 nucleotide sequences of many extant groups of Coleoptera, times as long as wide; pronotum widest at basal third and concluded that the Sphindidae is a family sharing a sister group with subexplanate sides narrower than antennal club; relationship with a clade comprising the superfamilies Tene- antennae shorter (not reaching posterior edge of pros- brionoidea and Lymexyloidea, while all cucujiformian superfamilies ternum), with thicker antennomeres, and nearly compact are presented in this interpretation as a intermixed aggregate. The and dorsoventrally compressed antennal club which is about list of contradictory interpretations (“phylogenies”) of the cucuji- twice as long as wide; setae of dorsum markedly not more formian beetles in current literature can be essentially continued. than twice as long as interstices between them. Fig. 12 .. .. Nevertheless, Buder et al. (2008) after comparison of different mo- ...... L. straminea sp. nov. lecular data concluded that “Two cucujoid families, i.e., the Silvani- dae (or at least Oryzaephilus) and Sphindidae, almost consistently Discussion form the basalmost clades of the entire cucujoid-tenebrionoid(- cleroid) assemblage”, placing the cleroids within mixture of cucu- Systematics joids and tenebrionoids. It is interesting to note that the cleroids (Peltidae and Trogossitidae) appeared in the fossil record much As demonstrated in the Comparison (see above), the new sub- earlier (Lower Jurassic) than both tenebrionoids (Mid Jurassic) and family bears a combination of characters which can scarcely cucujoids (Upper JurassiceLower Cretaceous) (Kirejtshuk and demonstrate a close relationship with any recent group in the Ponomarenko, 2013), i.e. the sequence of appearance of these su- family Sphindidae. In some sense the rank of this group is some- perfamilies in fossils is opposite to that in the conclusions of many what premature and only suggests the closest relatives for further recent neontologists. It is thought, therefore, that somewhat more elaboration of an equilibrium system of the superfamily. Sen Gupta probable approach to the real phylogeny will become possible only and Crowson (1979) regarded this family as a member of a basal after increasing of our knowledge on late Mesozoic cucujiformians. cucujoid branch and many authors prefer to consider it together The bionomics, larval development and ecology of the extinct with the Protocucujidae in a sister group relationship (McHugh, species of the new subfamily could be similar to those in recent 1993; Slipinski, 1999; Kirejtshuk, 2000; Leschen et al., 2005 etc.). groups, which seem to be exclusively associated with Myxomycota. The new subfamily could be a third member of this group. A more Discovering of five species in the same resource (Lebanese amber) detailed diagnosis and comparison took this factor into consider- suggests that resin-producing trees in the Lower Cretaceous “Leb- ation. Thus, the current placement of Libanopsinae subfam. nov. is anese” forest could be the target of regular infections by indicative of its closest relation to the recent subfamilies of Myxomycota. Sphindidae. Another possibility is to consider this new subfamily as equal to all recent groups combined as tribes in the subfamily On CLSM technique for the study of arthropod inclusions in amber (Sphindinae). However such changes of the family system or system within the larger cucujoid complex need a clearer definition of About ten years ago the confocal laser scanning microscopy the characters including whether they are plesio- or apomorphic. (CLSM) was accidentally found to be extremely appropriate for the This analysis is planned after further descriptions of some other study of amber inclusions (Böker and Brocksch, 2002). Various Lower Cretaceous groups of Cucujoidea. At this level of our ancient plant, insect and fungal samples preserved in amber have knowledge it is suspected that the unilobed maxillae and epipleura been studied using this technique (Clark and Daly, 2010; Speranza of Libanopsis species that steeply slope externally, their strongly et al., 2010). The application of CLSM methods for the study of in- transverse scutellum widening posteriorly with a long subtruncate clusions in amber is still not widespread, however in accordance apex and sparse erect setae on the dorsal integument seem to be with previous studies (cited above) our results show that this (aut) apomorphies. On the other hand, the tarsal formula 5-5-5 of method is rather promising, somewhat comparable with electron Libanopsinae subfam. nov. could be plesiotypic in comparison with microscopy and x-ray microtomography or sometimes in certain 4-segmented male metatarsi in all recent sphindid groups and aspects even surpassing them. The main advantages of the CLSM

Please cite this article in press as: Kirejtshuk, A.G., et al., Libanopsinae, new subfamily of the family Sphindidae (Coleoptera, Cucujoidea) from Lower Cretaceous Lebanese amber, with remarks on using confocal microscopy for the study of amber inclusions, Cretaceous Research (2014), http://dx.doi.org/10.1016/j.cretres.2014.02.008 18 A.G. Kirejtshuk et al. / Cretaceous Research xxx (2014) 1e19 method are: 1) comparable low price of study, 2) noninvasiveness, group here described. Besides, they thank Polina A. Kirejtshuk (the 3) effortlessness and speed of 3D-reconstructions, 4) capacity of first author’s daughter) for the assistance in preparing the illus- study of the same sample under both CLS and optic microscopes. trations for this paper. They are also grateful to the technicians at Amber is known to have a rather strong autofluorescence signal the Centre of Microscopy and Microanalysis of Saint-Petersburg at the exposure of blue laser (length of wave 405 nm). The cuticle of State University, namely N.S. Ovchinnikova and K.A. Benken, for many arthropods possesses a capacity to autofluorescence as well their advices concerning the CLSM technique. Important assistance (Valdecasas, 2008; Chetverikov, 2012 and papers cited therein). was received from George Poinar Jr. (Oregon State University), who According to our observations a rather distinct autofluorescent made the manuscript more fluent and transparent and also pro- signal can be recorded from the integument of small well preserved vided valuable comments. The study was partly carried out under insects in high quality amber inclusions examined under CLSM. the framework of the programme of the Presidium of the Russian Usually the bright background (autofluorescence of amber) and Academy of Sciences “Problems of the origin of life and formation of clear outlines of the studied inclusion are observed on the obtained the biosphere”. The first author is supported by the Russian Foun- CLSM images even if the amber is opaque and the inclusion is not dation of Basic Research (grant 12-04-00663a). This paper is a perfectly seen under conventional light microscope. Later the digital contribution to the team project “Biodiversity: Origin, Structure, CLSM data can be processed in graphic editors and impressive 3D Evolution and Geology” granted to DA by the Lebanese University. models obtained comparable with those obtained using micro- The authors greatly appreciate valuable comments on an earlier tomographic methods. When studying very old material or amber version of the manuscript received from anonymous reviewers. with numerous crevices, the autofluorescence may be completely absent because of a deep degradation of initial structure of insect cuticle and replacement of the latter by totally non-fluorescent References matter. Even in such cases, rarely it is possible to obtain good re- Azar, D., Perrichot, V., Néraudeau, D., Nel, A., 2003. New psychodid flies from the constructions of the insect surface using various modern filters Ò Ò Cretaceous ambers of Lebanon and France, with a discussion about Eophlebo- embedded in modern graphic editors e.g. Amira , Imaris or Fiji tomus connectens Cockerell, 1920 (Diptera, Psychodidae). Annals of the Ento- Open Source Image Processing Package (Schindelin et al., 2012). mological Society of America 96 (2), 117e127. e Results of CLSM study of amber inclusions drastically depends on Böker, C., Brocksch, D., 2002. Fascinating Amber. Carl Zeiss: Innovation 12, 20 23. Bouchard, P., Bousquet, Y., Davies, A.E., Alonso-Zarazaga, M.A., Lawrence, J.F., the medium used for mounting specimens. The samples prepared in Lyal, C.H.C., Newton, A.F., Reid, C.A.M., Schmitt, M., Slipinski, S.A., Smith, A.B.T., Canada Balsam can be studied under CLSM, but those mounted in 2011. Family-group names in Coleoptera (Insecta). ZooKeys 88, 1e972. http:// epoxy resin cannot, because the autofluorescence of the epoxy resin dx.doi.org/10.3897/zookeys.88.807. Buder, G., Grossmann, C., Hundsdoerfer, A., Klass, K.-D., 2008. A contribution to the totally masks the signal from the inclusion under examination. A phylogeny of the Ciidae and its relationships with other cucujoid and tene- similar observation has been recently achieved in the course of CLSM brionoid beetles (Coleoptera: Cucujiformia). Arthropod Systematics and Phy- study of the exoskeletons of eriophyoid mites (Asariformes, Erio- logeny 66 (2), 165e190. ’ Chetverikov, P.E., 2012. Confocal Laser Scanning Microscopy Technique for the Study phyoidea): the Hoyer s medium (includes Hum Arabic, chloralhy- of Internal Genitalia and External Morphology of Eriophyoid Mites (Acari: drate, glycerin and water) is the best choice for this case, but any Eriophyoidea). Zootaxa 3453, 56e68. media including the Iodine and polyvinyl chloride based Heinze Chetverikov, P.E., 2013. Comparative confocal microscopy of internal genitalia of phytoptine mites (Eriophyoidea, Phytoptidae): new generic diagnoses reflecting medium are not appropriate as it totally destroys (if Iodine media) or host-plant associations. Experimental and Applied Acarology 61 (2). http:// essentially diminishes (if Heinze medium) the quality of auto- dx.doi.org/10.1007/s10493-013-9734-2. fluorescent signal (Chetverikov, 2012, 2013; Chetverikov et al., 2012, Chetverikov, P.E., Beaulieu, F., Cvrkovic, T., Vidovic, B., Petanovic, R., 2012. Oziella sibirica (Eriophyoidea: Phytoptidae), a new eriophyoid mite species described 2013). Therefore using Canada Balsam for mounting the specimens using confocal microscopy and COI barcoding. Zootaxa 3560, 41e60. in amber for CLSM study is recommended as preferential. However Chetverikov, P.E., Cvrkovic, T., Vidovic, B., Petanovic, R.U., 2013. 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B.S., Reinhardt College, Waleska, Georgia, 1999. studied samples in amber (beetles), recent insects, acari and many University of Georgia (USA), Athens, 90 pp. http://athenaeum.libs.uga.edu/ other terrestrial and aquatic arthropods in dry or preserved media bitstream/handle/10724/6652/forrester_juanita_a_200305_ms.pdf?sequence¼1. Forrester, J.A., McHugh, J.V., 2007. A review of the Sphindidae (Coleoptera: Cucu- that were studied by previous authors (Valdecasas and Abad, 2011; joidea) of Madagascar. The Coleopterists Bulletin 61 (4), 590e603. Haug et al., 2011 and papers cited) demonstrate much stronger Haug, J.T.,Haug, C., Kutschera,V., Mayer,G., Maas, A., Liebau, S., Castellani, C., Wolfram,U., autofluorescence and resistance to photobleaching probably because Clarkson, E.N.K., Waloszek, D., 2011. Autofluorescence imaging, an excellent tool for comparative morphology. Journal of Microscopy 244 (3), 259e272. of the larger body-size and thickness of integuments. The photo- Hunt, T., Bergsten, J., Levkanicova, Z., Papadopoulou, A., St. John, O., Wild, R., bleaching of samples of the eriophyoid mites occurs over 40e60 s Hammond, P.M., Ahrens, D., Balke, M., Caterino, M.S., Gómez-Zurita, J., Ribera, I., even at low laser intensity (8e12%), but the samples of the beetles in Barraclough, T.G., Bocakova, M., Bocak, L., Vogler, A.P., 2008. A comprehensive fi phylogeny of beetles reveals the evolutionary origins of a superradiation. Sci- amber and dry or xed arthropods can be scanned by lasers with high ence 318, 1913e1916. http://dx.doi.org/10.1126/science.1146954 (on-line: Sci- intensity (35e50%) over 5e10 min without visible degradation of the ence 21 December 2007). quality of obtained images. This circumstance provides an opportu- Kirejtshuk, A.G., 2000. On origin and early evolution of the superfamily Cucujoidea nity to get images of high quality with lower rates of scanning and (Coleoptera, Polyphaga). Comments on the family Helotidae. The Kharkov Entomological Society Gazette 8 (1), 8e38. maximum resolution available in certain CLS microscope. Kirejtshuk, A.G., Azar, D., 2013. Current knowledge of Coleoptera (Insecta) from the Lower Cretaceous Lebanese amber and taxonomical notes for some Mesozoic e e Acknowledgements groups. Terrestrial Arthropod Reviews 6 (1 2), 103 134. http://dx.doi.org/ 10.1163/18749836-06021061. Kirejtshuk, A.G., Ponomarenko, A.G., 2013. Catalogue of fossil Coleoptera. Available The authors sincerely thank Adam S. Slipinski (CSIRO Ecosystem at: http://www.zin.ru/Animalia/Coleoptera/eng/paleosys.htm http://www.zin. ru/Animalia/Coleoptera/rus/paleosys.htm (accessed January 2013). Sciences, Division of Entomology, Canberra) and Alexey V. Kovalev Lawrence, J.F., Slipinski, A., Seago, A.E., Thayer, M.K., Newton, A.F., Marvaldi, A.E., (Zoological Institute of the Russian Academy of Sciences, St. 2011. Phylogeny of the Coleoptera based on morphological characters of adults Petersburg) for valuable discussion on attribution of the fossil and larvae. Annales Zoologici 61, 1e217.

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